Piperidine and pyrrolidine derivatives comprising a nitric oxide donor for treating stress

ABSTRACT

The present invention relates to chemical compounds comprising a nitric oxide (NO) donor and a superoxide ion (O 2   − ) scavenger, their preparation and their use in the treatment of conditions associated with oxidative stress or endothelial dysfunction. In particular, the present invention relates to chemical compounds of Formula (I) wherein: R 1  may be the same of different and are independently selected from hydrogen, alkoyl, carboxy, hydroxy, amino, amido, cyano, nitro, thio, sulphonyl, sulphoxide alkyl groups comprising an NO-donor, provided that at least one R 1  is a group comprising an NO-donor; R 2  may be the same or different and are independently selected from alkyl groups; n is an integer 1, 2, or 3; or a salt thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.09/600,711, filed Sep. 5, 2000, now U.S. Pat. No. 6,448,267 which is a371 of PCT/GB99/00231, filed on Jan. 22, 1999, which claims the benefitof United Kingdom 9801398.0, filed Jan. 22, 1998, the disclosures ofeach of which are incorporated herein by reference in their entirety.

The present invention relates to compounds capable of acting both assources of nitric oxide (NO-donors) and as scavengers of superoxide ion(O₂ ⁻) (superoxide dismutase (SOD)-mimetic).

Many disease states, including diabetes mellitus and variouscardiovascular diseases, are associated with oxidative stress andendothelial dysfunction. In particular, endothelial dysfunction is thehallmark of the pathophysiological process (atherosclerosis) leading toa spectrum of clinically related diseases of the cardiovascular system(e.g. from stable angina pectoris to myocardial infarction (MI) andcongestive heart failure), as well as being the primary event inpathologies relating to other body systems involving superoxide and/orother reactive oxygen species (ROS).

For over a century, nitroglycerin (GTN) has been the drug of choice forthe treatment of various types of myocardial ischemia (angina),including myocardial infarction, and a mainstay in the treatment ofother heart diseases with and without ischemic etiology (congestiveheart failure, isolated systolic and resistant hypertension). Because ofits pathogenic nature (chronicity with acute exacerbation), prophylacticand acute treatments are necessary to prevent complications withpotentially fatal outcomes (>25% death for acute MI).

However, the phenomenon of tolerance to the anti-anginal effects of GTNand to all other existing organic nitrates is of a special clinicalsignificance. In particular, early development of tolerance to the drugis by far the most serious drawback of nitrate therapy, especiallyduring acute myocardial infarction.

Evidence has been provided to support an involvement of the superoxideanion in the mechanism/s underlying nitrate tolerance andcross-tolerance (Munzel et al., J. Clin. Invest. 95, 187-194 (1995)).According to this report, increased levels of superoxide anion werefound to accompany tolerance development to GTN in vascular tissue afterin vivo administration of the drug. Treatment with superoxide dismutase(SOD) significantly enhanced maximal relaxation of control and tolerantvascular tissue to GTN and other exogenous and endogenous vasodilators.This can be explained on the basis of an enhancing effect of superoxideon the induction of tolerance (pseudo-tolerance). It is believed that ifthe normally tightly controlled balance between nitric oxide (NO) andsuperoxide (O₂ ⁻) in the vascular wall is disturbed, elevated levels ofsuperoxide anion prevail, inactivating NO and furthermore generatingtoxic peroxynitrite. The protective effect of superoxide dismutaseagainst nitrate tolerance has been demonstrated in vitro (Jia et al., J.Pharmacol. Exp. Ther., 267,371-378 (1993) and Kowaluk et al., J.Pharmacol. Exp. Ther., 255, 109-144 (1990)). However, being an enzyme,SOD is practically cell impermeable and cannot therefore be usedtherapeutically. There remains therefore a need for a nitrate-basedtherapy which does not suffer from the problem of nitrate tolerance.

According to the present invention there is provided a chemical compoundcomprising a nitric oxide (NO) donor and a superoxide (O₂ ⁻) scavenger.

The nitric oxide donor may comprise any group capable of acting as asource of nitric oxide (NO). Preferably, the nitric oxide donor is an—ONO₂ group. The superoxide scavenger may comprise any group capable ofacting as a scavenger of superoxide (O₂ ⁻). Preferably, the superoxidescavenger is a nitroxide free radical (N→O.) group.

The compounds of the present invention may comprise one or more NOdonors and one or more superoxide scavengers.

The compounds of the present invention not only provide a source ofnitric oxide but in acting as an antioxidant scavenger of superoxideanion give rise to both a direct benefit derived from removal ofinjurious superoxide anion and a benefit in protecting both ambient andendogenous and liberated exogenous NO from inactivation by superoxideanion.

Without prejudice to the scope of the present invention, it is believedthat oxidative damage is mediated by intracellular redox-active metalreactions catalyzed by highly reactive oxygen species (i.e. hydroxylradicals). The generation of such reactive oxygen products depends onthe availability of their common precursor, the superoxide anion.Mitochondria, microsomes and other various enzyme systems are known toproduce superoxide anion that reacts with nitric oxide at or neardiffusion controlled rates to form the powerful oxidant peroxynitrite.At pH 7.4, peroxynitrite protonates to form peroxynitrous acid (pKa 6.6)which decays homolytically to form hydroxyl and nitrogen dioxideradicals in addition to a host of other ions. The extent to which theselater reactive ions and radicals can cause cellular damage and deathdepends on the rate of formation of their peroxynitrite precursor. Undercontrol (non-diseased) conditions, where the levels of NO are found inequilibrium between rates of NO-synthesis and degradation, the rate offormation of this peroxynitrite precursor is thought to depend solely onthe levels of the superoxide anion. This is particularly important incases involving “below-normal” levels of “biologically active” NO, suchas in diseases for which therapeutic intervention with exogenousNO-donors is clinically indicated. Consequently, the extra- andintra-cellular activity of the enzyme SOD must have a cardinal role inmaintaining cellular survival, tissue integrity and adequately balancedphysiological function.

Since superoxide anion is an available and continuously-formedby-product generated through normal metabolic processes, and since itselimination is mediated either by dismutation by the enzyme SOD or viaits reaction with NO to form the potentially hazardous peroxynitrite, itis now believed that the ultimate means by which a modification and/ortreatment of ‘pathological processes’ involving imbalanced ratio of NOto superoxide is by an intervention with therapeutic agents capable ofsimultaneously and favourably affecting both components; the NO and O₂⁻.

By virtue of the NO donor and superoxide scavenging activities beingphysically linked in the same molecule, the compounds of the presentinvention ensure that an increase in the level of NO is accompanied byreduced levels of superoxide thereby avoiding high levels ofperoxynitrite and oxidant metabolites thereof. Preferred compoundsaccording to the present invention are of the formula:

Preferred compounds according to the present invention are of theformula:

wherein R¹ may be the same or different and are independently selectedfrom hydrogen, alkoyl, alkoxy, carboxy, hydroxy, amino, amido, cyano,nitro, thio, sulphonyl, sulphoxide, alkyl groups and groups comprisingan NO-donor, provided that at least one R¹ is a group comprising anNO-donor group;

R² may the same or different and are independently selected from alkylgroups;

n is an integer 1, 2 or 3;

or a salt thereof.

The groups R¹ may be the same or different and are independentlyselected from hydrogen, alkoyl, alkoxy, carboxy, hydroxy, amino, amido,cyano, nitro, thio, sulphonyl, sulphoxide, alkyl groups and groupscomprising an NO-donor group, provided that at least one R¹ is a groupcomprising an NO-donor. R¹ groups which do not comprise an NO-donorgroup are preferably hydrogen or alkyl groups. Each R¹ group comprisingan NO-donor group may comprise one or more NO-donor groups. Preferably,each NO-donor group is an —ONO₂ group.

Each R¹ group comprising an NO-donor group may comprise an NO-donorgroup alone or may comprise an NO-donor group linked via a C₁ to C₂₀alkylene chain optionally comprising one or more heteroatoms. Thealkylene chain may be branched or unbranched, cyclic or acyclic,saturated or unsaturated, where cyclic the alkylene chain is preferablyC₃ to C₁₂, more preferably C₅ to C₁₀, more preferably C₅ to C₇. Whereacyclic, the alkylene chain is preferably C₁ to C₁₆, more preferably C₁to C₆. The alkylene chain may be unsubstituted or substituted as definedhereinbelow in respect of alkyl groups. The alkylene chain may compriseone or more heteroatoms, for example nitrogen, oxygen or sulphur atomswhich may interrupt the alkylene chain or may link the alkylene chain tothe NO-donor group or to the rest of the molecule (such that the R¹group comprising the NO-donor group is linked to the rest of themolecule via a heteroatom, such as an oxygen (e.g. ether linkage),nitrogen (e.g. amino or amido linkage) or sulphur (e.g. thioetherlinkage)).

Preferred R¹ groups comprising NO-donors comprise groups of the formula:

—X—Y—ONO₂

where X is absent or is O, NH or S; and

Y is a C₁₋₂₀ alkylene chain

Preferably, X is O, NH or S.

In a preferred embodiment, at least one R¹ is a polar or ionisable groupsuch as carboxyl or amino, which improves the solubility of thecompound.

The groups R² may be the same or different and are selected from alkylgroups. Preferably, the groups R² are each methyl groups. In analternative preferred embodiment, one or more of the groups R² is alipophilic group, such as a substituted or unsubstituted C₁ to C₈ alkylgroup, capable of assisting in transport of the compound across theblood-brain membrane.

n may be 1, 2 or 3. Preferably n is 1 or 2, More preferably n is 2.

As used here reference to alkyl groups means a branched or unbranched,cyclic or acyclic, saturated or unsaturated (e.g. alkenyl or alkynyl)hydrocarbyl radical. Where cyclic, the alkyl group is preferably C₃ toC₁₂, more preferably C₅ to C₁₀, more preferably C₅ to C₇. Where acyclic,the alkyl group is preferably C₁ to C₁₆, more preferably C₁ to C₆, morepreferably methyl. Reference in the present specification to an alkoxygroup means an alkyl-O— group. Reference to alkoyl group means analkyl-CO— group.

The alkyl groups may be substituted or unsubstituted, preferablyunsubstituted. Where substituted, there will generally be 1 to 3substituents present, preferably 1 substituent. Substituents may includehalogen atoms and halomethyl groups such as CF₃ and CCl₃; oxygencontaining groups such as oxo, hydroxy, carboxy, carboxyallyl, alkoxy,alkoxy, alkoyl, alkoyloxy, aryloxy, aryloyl and aryloyloxy; nitrogencontaining groups such as amino, amido, alkylamino, dialkylamino, cyano,azide, nitrato and nitro; sulphur containing groups such as thiol,alkylthiol, sulphonyl and sulphoxide; heterocyclic groups containing oneor more, preferably one, heteroatom, such as thienyl, furanyl, pyrrolyl,imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl pyrrolidinyl,pyrrolinyl, imidazolidinyl, imidazolinyl, pyrazolidinyl,tetrahydrofuranyl, pyranyl, pyronyl, pyridyl, pyrazinyl, pyridazinyl,benzofuranyl, isobenzofuryl, indolyl, oxyindolyl, isoindolyl, indazolyl,indolinyl, 7-azaindolyl, isoindazolyl, benzopyranyl, coumarinyl,isocoumarinyl, quinolyl, isoquinolyl, naphthridinyl, cinnolinyl,quinazolinyl, pyridopyridyl, benzoxazinyl, quinoxadinyl, chromenyl,chromanyl, isochromanyl and carbolinyl; alkyl groups, which maythemselves be substituted; and aryl groups, which may themselves besubstituted, such as phenyl and substituted phenyl. Alkyl includessubstituted and unsubstituted benzyl.

Reference to amino includes substituted or unsubstituted amino.

Reference in the present specification to halogen means a fluorine,chlorine, bromine or iodine radical, preferably fluorine or chlorineradical.

The present invention extends to dimers and higher multimers of thecompounds of the present invention; for example dimers of compounds ofthe present invention linked via R¹ groups (e.g. via an alkylene chaintherein).

Particularly preferred compounds according to the present inventioncomprise:

4-Nitrato-2,2,6,6-tetramethylpiperidinyloxy, free radical(TEMPO-4-mononitrate)

3,4-Dinitrato-2,2,6,6-Tetramethylpiperidinyloxy, free radical(TEMPO-3,4-dimitrate)

3,4,5-Trinitrato-2,2,6,6-Tetramethylpiperidinyloxy, free radical(TEMPO-3,4,5-trinitrate)

4(2,3-Dinitrato-Prop-1-oxy)2,2,6,6-Tetramethyl-piperidinyloxy, freeradical (4-(2,3-dinitrato-prop-1-oxy) TEMPO)

1,3-di (4Oxy-TEMPO)-2-Nitrato-Propane

1,4-di (4-Oxy-TEMPO)-2,3-Dinitrato-Butane

3-Nitrato-4-carboxy-2,2,6,6-tetramethylpiperidinyloxy, free radical(TEMPO-4-carboxyl-3-nitrate)

4Nitrato-3-carbamoyl-2,2,5,5-tetramethyl-3-pyrrolidin-1-yloxy, freeradical (PROXYL-3-carbamoyl-4-nitrate)

The present invention further extends to methods of synthesising thecompounds of the present invention. The compounds of the presentinvention may be synthesised according to the following generalisedreaction schemes. It will be appreciated that te reaction schemes aremerely illustrative of generally applicable procedures which may bemodified as appropriate to produce the compounds of the presentinvention. In the following reaction schemes the 2,2,6,6-tetrasubstitution present in, for example TEMPO compounds is, for simplicity,not shown.

According to a further aspect of the present invention there is provideda compound according to the present invention for use in a method oftreatment.

The compounds of the present invention may be employed in the treatmentof any condition associated with endothelial dysfunction or oxidativestress including diabetes mellitus, cardiovascular diseases (such asischaemic heart disease, angina pectoris, myocardial infarction,congestive heart failure, atherosclerosis, hypertension and arrhythmia),asthma, trauma, shock (hypovolumic, neurogenic or septic),neurotoxicity, neurodegenerative and neurological disorders (includingAlzheimer and Parkinson's diseases, amyotrophic lateral sclerosis,multiple sclerosis, convulsive (seizure) disorders, AIDS-dementia anddisorders which involve processes of learning and memory), disorders ofgastric secretions, relaxation and peristalsis of the intestinal tract(including sphincters), drug and disease-induced nephropathies,pathological (premature) and physiological uterine contractions,cellular defense impairment, endothelial dysfunction-induced diseasesand insulin-resistance in diabetes, pregnancy-induced hypertension,chemotaxis and phagocytic impairment in immunological disorders,cerebrovascular diseases, aggregation disorders, fertility andreproductive disorders (e.g. penile erection and treatment of maleimpotence).

According to a further aspect of the present invention there is provideduse of a compound according to the present invention for use in themanufacture of a medicament for treating a condition associated withoxidative stress or endothelial dysfunction, preferably diabetesmellitus or cardiovascular disease.

According to a further aspect of the present invention there is provideda method of treating a disease associated with oxidative stress orendothelial dysfunction (such as diabetes mellitus or cardiovasculardisease) comprising administering to a patient in need of such treatmentan effective dose of a compound of the present invention

According to a further aspect of the present invention there is provideda pharmaceutical composition comprising a compound of the presentinvention in combination with a pharmaceutically acceptable excipient.

Compounds of the present invention may be administered in a formsuitable for oral use, for example a tablet, capsule, aqueous or oilysolution, suspension or emulsion; for topical use including transmucosaland transdermal use, for example a cream, ointment, gel, aqueous or oilsolution or suspension, salve, patch, plaster or as a component of alubricant for a condom; for nasal use, for an example a snuff, nasalspray or nasal drops; for vaginal or rectal use, for example asuppository; for administration by inhalation, for example a finelydivided powder or a liquid aerosol; for sub-lingual or buccal use, forexample a tablet or capsule; or for parenteral use (includingintravenous, subcutaneous, intramuscular, intravascular or infusion),for example a sterile aqueous or oil solution or suspension, orincorporated in a biodegradable polymer. In general the abovecompositions may be prepared in a conventional manner using conventionexcipients, using standard techniques well known to those skilled in theart of pharmacy. Preferably, the compound is administered orally ortopically.

For oral administration, the compounds of the invention will generallybe provided in the form of tablets or capsules or as an aqueous solutionor suspension.

Tablets for oral use may include the active ingredient mixed withpharmaceutically acceptable excipients such as inert diluents,disintegrating agents, binding agents, lubricating agents, sweeteningagents, flavouring agents, colouring agents and preservatives. Suitableinert diluents include sodium and calcium carbonate, sodium and calciumphosphate, and lactose, while corn starch and alginic acid are suitabledisintegrating agents. Binding agents may include starch and gelatin,while the lubricating agent, if present, will generally be magnesiumstearate, stearic acid or talc. If desired, the tablets may be coatedwith a material such as glyceryl monostearate or glyceryl distearate, todelay absorption in the gastrointestinal tract.

Capsules for oral use include hard gelatin capsules in which the activeingredient is mixed with a solid diluent, and soft gelatin capsuleswherein the active ingredient is mixed with water or an oil such aspeanut oil, liquid paraffin or olive oil.

For intramuscular, intraperitoneal, subcutaneous and intravenous use,the compounds of the invention will generally be provided in sterileaqueous solutions or suspensions, buffered to an appropriate pH andisotonicity. Suitable aqueous vehicles include Ringer's solution andisotonic sodium chloride. Aqueous suspensions according to the inventionmay include suspending agents such as cellulose derivatives, sodiumalginate, polyvinyl-pyrrolidone and gum tragacanth, and a wetting agentsuch as lecithin. Suitable preservatives for aqueous suspensions includeethyl and n-propyl p-hydroxybenzoate. The compounds of the invention mayalso be provided in a biodegradable polymer, for example for use inconjunction with stints in angioplasty (e.g. adsorbed on a stent orapplied directly to the site of the procedure for slow release of theactive agent).

It will be appreciated that the dosage levels used may vary over quite awide range depending upon the compound used, the severity of thesymptoms exhibited by the patient and the patient's body weight. Withoutlimitation to the present invention, typical dosages for example for thetreatment of angina may be of the order of 1 to 100 mg, preferably 5 to40 mg, given two or three times daily or 1 to 200 mg, preferably 20 to50 mg, in sustained release formulations given once or twice daily.Typical dosages for example for acute myocardial infarction may be ofthe order of 0.1 to 10 mg, preferably 1 to 2 mg, sublingually; 0.5 to 50mg, preferably 5 to 10 mg, orally; or 1 to 100 micrograms, preferably 10to 20 micrograms, per minute intravenously.

BRIEF DESCRIPTION OF DRAWINGS.

The invention will now be described with reference to the figures inwhich:

FIG. 1 illustrates inhibition by TEMPO-4-mononitrate of reduction offerricytochrome C by a xanthine oxidase/hypoxanthine superoxide aniongenerating system in vitro. Values are mean±s.e.m.

FIG. 2 illustrates relaxation response curves of control RARs to GTN andNO-TEMPO;

FIG. 3 illustrates relaxation response curves of control RARs to SNAP(n=8) and NO-TEMPO (n=11);

FIG. 4 illustrates relaxation response curves to GTN of control andGTN-tolerant RARs;

FIG. 5 illustrates relaxation response curves to NO-TEMPO of control(n=11) and GTN tolerant (n=7) RARs;

FIG. 6 illustrates relaxation response curves of NO-TEMPO-pretreatedRARs (0.44 mM of NO-TEMPO) to GTN and NO-TEMPO;

FIGS. 7 illustrates relaxation response curves to NO-TEMPO before andafter pretreatment of RARs with 0.44 mM of NO-TEMPO; and

FIG. 8 illustrates relaxation response curves of RARs (n=8) to GTNbefore and after pretreatment with 0.44 mM of NO-TEMPO.

It will be appreciated that the invention is described by way of exampleonly, and that modifications of detail may be made without departingfrom the scope of the present invention.

EXPERIMENTAL Synthesis of Compounds

4-Nitrato-2,2.6,6-tetramethylpiperidinyloxy, free radical(TEMPO-4-mononitrate)—(Reaction Scheme 1)

The title compound was prepared by nitration of commercially available4-hydroxy-TEMPO. 4-Hydroxy-TEMPO (Aldrich Chemical Co.) (20 mmol) wasadded portionwise to a cooled (0-5° C.) mixture of filming nitric acid(3 ml) and concentrated sulphuric acid (3 ml). After addition wascomplete, the mixture was brought to room temperature and stirredvigorously for a further 15 minutes. The mixture was added continuouslyand dropwise to a cooled (5° C.) mixture ofdiethylether:acetonitrile:water (150 ml:50 ml:25 ml) with vigorousstirring. Sodium chloride (5 g) was added to facilitate separation ofthe organic layer, which was then evaporated to dryness under reducedpressure. The residue was dissolved in diethyl ether (50 ml) and washedtwice with ice-water (20 ml) and once with 2% aq. potassium carbonate(20 ml) before being dried (MgSO4) and evaporated to dryness.

Alternatively, nitration of 4-hydroxy-TEMPO (and nitration of all otherhydroxy precursors described by this invention) can be easily achievedby direct exposure of the compound to the gaseous nitrating agentdinitrogen tetroxide (N₂O₄). Here, N₂O₄ (1.25 equivalents) wasintroduced to the desired weight into a tarred, precooled (−20° C.)round-bottom flask containing 50 ml of dry tetrahydrofurane-hexanemixture (1:1). 4-Hydroxy-TEMPO (1 equivalent) dissolved in drytetrahydrofurane (25 ml, −20° C.) was added dropwise into the nitratingsolution with stirring. The reaction temperature was maintained below−10° C. during the entire addition period, after which it was left torise to room temperature within 1 hr. Nitrogen gas was bubbled into thereaction mixture until no more red gas is seen to exit in the upper partof the flask. The organic solvents were evaporated to near dryness andthe residue dissolved in methyl tert-butyl ether (100 ml), washed oncewith water (50 ml) and twice with 50 ml ice-cold 2% sodium bicarbonatesolution. The ether was separated, dried (MgSO₄) and evaporated todryness.

The red residue was purified by column chromatography using silica gel(60) and eluted with diethyl ether: petroleum ether (20:80) to furnishthe title compound in 90-95% yield. The identity of the product wasconfirmed by NMR and ESR spectroscopy and by TLC analysis by reactionwith diphenylamine, which after heating to 80° C. for 1 minute, gives adeep green colour typical of nitrated nitroxides.

Alternatively, the title compound was prepared by dissolving 3.44 g (20mmol) of 4-hydroxy-TEMPO in 50 ml of dichloromethane. The mixture wascooled on ice bath and 6 ml of thionyl bromide in 25 ml ofdichloromethane was added dropwise during 30 min. After completion thereaction was stirred for an additional 3 hours during which it reachedroom temperature. The solvent was washed successively with water (75 ml)and 10% sodium carbonate solution (75 ml), dried (MgSO₄) and evaporatedto dryness. The residue was chromatographed on silica gel and elutedwith petroleum ether: dichloromethane (80:20) to furnish 4.71 g (94%) ofpure 4-bromo-TEMPO. The product was dissolved in 50 ml of acetonitrilecontaining 4.25 g (25 mmol) of argentum nitrate. The reaction mixturewas heated at 60° C. for 4 hours. The precipitate was removed byfiltration and washed with 30 ml of cold acetonitrile. The combinedfiltrate was removed to dryness under reduced pressure and separated bychromatography as described above to furnish 4.2 g (87%) of the titlecompound.

The following compounds were prepared using similar procedures and inaccordance with the Reaction Schemes set out above:

3,4-Dinitrato-2,2,6,6-Tetramethylpiperidinyloxy, free radical(TEMPO-3,4-dinitrate)

3,4,5-Trinitrato-2,2,6,6-Tetramethylpiperidinyloxy, free radical(TEMPO-3,4,5-trinitrate)

4-(2,3-Dinitrato-Prop-1-oxy)-2,2,6,6-Tetramethyl-piperidinyloxy, freeradical (4-(2,3-dinitrato-prop-l-oxy) TEMPO)

1,3-di (4-Oxy-TEMPO)-2-Nitrato-Propane

1,4-di (4-Oxy-TEMPO)-2,3-Dinitrato-Butane

3-Nitrato-4-carboxyl-2,2,6,6-tetramethylpiperidinyloxy, free radical(TEMPO-4-carboxyl-3-nitrate)

3,4-Dinitrato-2,2,6,6-tetramethylpiperidinyloxy, free radical(TEMPO-3,4-dinitrate)—Reaction Scheme 2)

To 4.26 g (25 mmol) of 4-oxo-2,2,6,6-tetramethyl-1-piperidinyloxy, freeradical (4-oxo-TEMPO, Aldrich Chemical Co.) dissolved in 50 ml of carbontetrachloride (CCL₄) there was added portion wise 4.895 g (27.5 mmol) ofdried, fine powder of N-bromosuccinimide over 25 min with stirring. Thereaction was left overnight and filtered. The precipitate was washedwith 20 ml of CCl₄ and the combined filtrate evaporated to dryness underreduced pressure. The red oil residue (which solidifies to a waxyproduct upon standing for one day) was chromatographed on silica gel andeluted with chloroform:hexane (60:40) to furnish 5.42 g (87%) of3-bromo-4-oxo-TEMPO. The product was dissolved in 50 ml of dry diethylether and added in one portion to ice-cold 25 ml of 1.0M solution oflithium aluminum hydride in diethyl ether. After stirring for 30 min onice bath, 25 ml of methanol was added dropwise to destroy excesshydride.

The solvent was filtered and evaporated to dryness. The crude residuewas dissolved in dichloromethane (50 ml) and treated for 5 hours withthionyl bromide (6 ml) at room temperature. The reaction mixture waswashed twice with equal volumes of water and twice with 5% sodiumcarbonate solution and organic phase separated and dried on magnesiumsulfate. After removing the solvent under reduced pressure, the desireddibromo derivative was isolated by column chromatography (silica gel 60)after elution with petroleum ether. The red oily product was dissolvedin 50 ml of acetonitrile containing 30 mmol argentum nitrate andnitration and separation of the desired dinitrated derivative performedas described above for the mononitrated TEMPO.

3,4,5-Trinitrato-2,2,6,6-tetramethylpiperidinyloxy, free radical(TEMPO-3,4,5-trinitrate)—(Reaction Scheme 3)

To 4.26 g (25 mmol) of 4-oxo-2,2,6,6-tetramethyl-1-piperidinyloxy, freeradical (4oxo-TEMPO, Aldrich Chemical Co.) dissolved in 150 ml of carbontetrachloride (CCl₄) there was added portion wise 98 g (55 mmol) ofdried, fine powder of N-bromosuccinimide over 25 min with stirring. Thereaction was left overnight and filtered. The precipitate was washedwith 25 ml of CCl₄ and the combined filtrate evaporated to dryness underreduced pressure. The red oil residue was chromatographed on silica geland eluted with chloroform:hexane (30:70) to furnish 6.75 g (82%) of3,5-dibromo-4-oxo-TEMPO. The product was dissolved in 100 ml of drydiethyl ether and added in one portion to ice-cold 50 ml of 1.0Msolution of lithium aluminum hydride in diethyl ether. After stirringfor 20 min on ice bath, 25 ml of methanol was added dropwise to destroyexcess hydride.

The solvent was filtered and evaporated to dryness. The crude reside wasdissolved in dichloromethane (50 ml) and treated for 5 hours withthionyl bromide (6 ml) at room temperature. The reaction mixture waswashed twice with equal volumes of water and twice with 5% sodiumcarbonate solution and organic phase separated and dried on magnesiumsulfate. After removing the solvent under reduced pressure, the desiredtribromo derivative was isolated by column chromatography (silica gel60) after elution with heptane. The red oily product was dissolved in150 ml of acetonitrile containing 0.1 M argentum nitrate and nitrationand separation of the desired dinitrated derivative performed asdescribed above for the mono and dinitrated TEMPO.

4-(2,3-Dinitrato-prop-1-oxy)-2,2,6,6-tetramethylpiperidinyloxy, freeradical (4-[2,3-dinitrato-prop-1-oxy]-TEMPO)—(Reaction Scheme 4)

4.3g of 4-hydroxy-TEMPO (25 mmol) was dissolved in 50 of drytetrahydrofuran (THF) and cooled on ice bath. 0.66 g (27.5 mmol) ofmetal sodium was added in one portion and the reaction mixture stirredvigorously for 40 min. 25 ml of THF containing 3.76 g (27.5 mmol) ofeipibromohydrin was added dropwise with stirring at room temperature forone hour. The reaction mixture was evaporated to dryness and residuetreated with 20% sodium hydroxide solution for 3 hours at 55° C. Aftercooling, the reaction mixture was extracted 3 times with 50 ml each ofethyl acetate. The organic layer was separated and the combined extractsdried and evaporated to dryness. The viscous oily residue was added to acooled (0° C.) mixture of fuming nitric acid and sulfuric acid (3.7 mleach) and stirred for 30 min. The reaction mixture was poured in 1 mlportions into a 300 ml of cold water: acetonitrile:diethyl ether mixture(10:20:70) and stirred for 10 min while nitrogen gas was bubbled intothe stirred solution. The organic phase was separated, washed with waterand 5% sodium carbonate solution, dried over magnesium sulfate andevaporated to dryness. The reddish oily residue was separated oversilica gel and eluted with chloroform to yield 5.1 g (61% based on4-hydroxy-TEMPO) of the desired product4-(2.3-Dinitrato-prop-1-oxy)-2,2,6,6-tetramethylpiperidinyloxy, freeradial (4-[2,3-dinitrato-prop-1-oxy]-TEMPO).

1,3-Di-(4-oxy-TEMPO)-2-nitratopropane—(Reaction Scheme 5)

8.6 g (50 mmol) of 4-hydroxy-TEMPO were dissolved in 80 ml of drytetrahydrofuran (THF) and cooled on ice bath. 1.44 g (60 mmol) of metalsodium was added in one portion and the reaction mixture stirredvigorously for 40 min. 25 ml of THF containing 5.4 g (25 mmol) ofdibromoacetone was added dropwise with stirring at room temperature forone hour. The reaction mixture was cooled on ice-water bath and 30 ml of1.0 M solution of lithium aluminum hydride in diethyl ether was added inone portion. After stirring for 20 min on ice bath, 25 ml of methanolwas added dropwise to destroy excess hydride. The solvent was filteredand evaporated to dryness. The viscous oily residue was added to acooled (0° C.) mixture of fuming nitric acid and sulfuric acid (3.7 mleach) and stirred for 30 min. The reaction mixture was poured in 1 mlportions into a 300 ml of cold water: acetonitrile:diethyl ether mixture(10:20:70) and stirred for 10 min while nitrogen gas was bubbled intothe stirred solution. The organic phase was separated, washed with waterand 5% sodium carbonate solution, dried over magnesium sulfate andevaporated to dryness. The reddish oily residue was separated oversilica gel and eluted with dichloromethane:diethyl ether (80:20) toyield 6.45 g (57% based on 4-hydroxy-TEMPO) of the desired product1,3-Di-(4-oxy-TEMPO)-2-nitratopropane.

1,4-Di-(4-oxo-TEMPO)-2.3-dinitratobutane—(Reaction Scheme 6)

8.6 g (50 mmol) of 4-hydroxy-TEMPO were dissolved in 80 ml of drytetrahydrofuran (THF) and cooled on ice bath. 1.44 g (60 mmol) of metalsodium was added in one portion and the reaction mixture stirredvigorously for 40 min 25 ml of THF containing 2.15 g (25 mmol) of(±)-1,3-butadiene diepoxide was added dropwise with stirring at roomtemperature for five hours. The reaction mixture was filtered andevaporated to dryness. The viscous oily residue was added to a cooled(0° C.) mixture of fuming nitric acid and sulfinic acid (5.8 ml each)and stirred for 30 min. The reaction mixture was poured in 1 ml portionsinto a 300 ml of cold water:acetonitrile:diethyl ether mixture(10:20:70) and stirred for 10 min while nitrogen gas was bubbled intothe stirred solution. The organic phase was separated, washed with waterand 5% sodium carbonate solution, dried over magnesium sulfate andevaporated to dryness. The reddish oily residue was separated oversilica gel and eluted with dichloromethane:diethyl ether (60:40) toyield 5.59 g (43% based on 4-hydroxy-TEMPO) of the desired product1,4-Di-(4-oxo-TEMPO)-2.3-dinitratobutane.

4-Nitrato-3-carbamoyl-2,2,5,5-tetramethyl-3-pyrrolidin-1-yloxy, freeradical (PROXYL-3-carbamoyl-4-nitrate)—(Reaction Scheme 7)

3.6 g (20 mmol) of 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrrolin-1-yloxy,free radical commercially available from Aldrich Chemical Co. was addedportion wise to 6 ml of cool concentrated hydrobromic acid solutioncontaining 50 mg of 1,1′-azobis (cyclohexanecarbonitrile) and stirredovernight. The resulting solution was diluted with 14 ml of ice coldwater and extracted 5 times each of 50 ml of ether. The combined etherextracts was washed twice with water and twice with 30 ml each of 5%sodium carbonate solution. The organic solvent was dried (sodiumsulfate) and evaporated to dryness. The resulting crude 4bromo-3-carboxyderivative was dissolved in 50 ml of acetonitrile, 6 g of argentumnitrate were added and the solution refluxed for 2 hrs. After cooling toroom temperature, the inorganic salts were removed by filtration and theorganic solvent removed. The residue was triturated with a mixture ofhexane-ether solution (1:1) and the solvent removed The residue was thenseparated by flash chromatography (dichloromethane:ethyl acetate 4:1) togive the pure title compound.

3-Nitratomethyl-2,2,5,5-tetramethylpyrrolidinyloxy, free radical(3-nitratomethyl-PROXYL)

To 4.26 g (25 mmol) of 4-oxo-2,2,6,6-tetramethyl-1-piperidinyloxy, freeradical (4-oxo-TEMPO) dissolved in 50 ml of carbon tetrachloride (CCl₄)there was added portion wise 4.895 g (27.5 mmol) of dried, fine powderof N-bromosuccinimide over 25 min with stirring. The reaction was leftovernight and filtered. The precipitate was washed with 20 ml of CCl₄and the combined filtrate evaporated to dryness under reduced pressure.The red oil residue (which solidifies to a waxy product upon standingfor one day) was chromatographed on silica gel and eluted withchloroform:hexane (60:40) to furnish 5.42 g (87%) of3-bromo-4-oxo-TEMPO. The product was dissolved in 20 ml of methanol andadded dropwise to 30 ml of ice-cold, freshly prepared solution of sodiummethoxide (60 mmol). After addition is complete, the reaction mixture isstirred at room temperature for 5 hours at the end of which the reactionis brought to reflux for 30 min. After cooling, the solvent was filteredand evaporated to dryness under reduced pressure. The residue wasdissolved in 50 ml of dry diethyl ether and added dropwise to 30 ml of1.0 M solution of lithium aluminum hydride in diethyl ether. Afterstirring for 20 min on ice bath, 25 ml of methanol was added dropwise todestroy excess hydride. The solvent was filtered and evaporated todryness. The viscous oily residue was added to a cooled (0° C.) mixtureof fuming nitric acid and sulfric acid (3.7 ml each) and stirred for 30min. The reaction mixture was poured in 1 ml portions into a 300 ml ofcold water:acetonitrile:diethyl ether mixture (10:20:70) and stirred for10 min while nitrogen gas is bubbled into the stirred solution. Theorganic phase was separated, washed with water and 5% sodium carbonatesolution, dried over magnesium sulfate and evaporated to dryness. Theyellow-orange oily residue was separated over silica gel and eluted withdichloromethane:hexane (80:20) to yield the title compound3-nitratomethyl-2,2,5,5-tetramethylpyrrolidinyloxy, free radical(3-nitratomethyl-PROXYL) in 63% yield.

Biological Testing

Assessment of superoxide scavenging activity in vitro

The superoxide scavenging activity of TEMPO-4-mononitrate (0.003-3 mM)and reference compound superoxide dismutase (SOD, 200 U/ml), weredetermined using a photometric microassay in vitro (Laight et al.,Environ. J. Toxicol. Pharmracol., 3, 65-68, 1997) developed from theassay of McCord & Fridovich (J. Biol. Chem., 244, 6059-55, 1969). Theassay mixture consisted of (final concentration): 50 μl ferricytochromec (100 μM); 10 μl xanthine oxidase (20 mU/ml); 20 μl hypoxanthine (100μM); and 20 μl sample dissolved in isotonic phosphate (10 mM)-bufferedsaline (PBS, pH 7.4) to make a total volume of 100 μl in a 96-wellplate. The reaction was conducted at room temperature and initiated bythe addition of hypoxanthine. The increase in optical density (OD) at500 nm was measured over a 3 min period of 30 s intervals and initialreaction rates determined at least in triplicate.

There was a linear relationship between the initial reaction rate andthe concentration of xanthine oxidase (5-20 mU/ml) (r=0.9982, P<0.002,n=4). Blank controls lacking either enzyme or substrate showed noactivity. Xanthine oxidase at 20 mU/rml, which provided an initialreaction rate of 28.3±1.7 mOD/min (n=4), was adopted for subsequentinhibition studies (see McCord & Fridovich, 1969). The initial reactionrate was depressed by SOD (200 U/ml) by 95.3±1.1% (n=4, P<0.01) and byTEMPO-4-mononitrate (see FIG. 1). TEMPO-4-mononitrate exhibited a pIC₅₀value of 2.89±0.03 (n=5,p<0.001).

Vasorelaxation and Tolerance-Inducing Properties

Materials: Male Sprague-Dawley rats (250-300 g) were purchased from theAnimal Care Facility of the Hebrew University and used in the rat aorticrings (RARs) relaxation studies in vitro and the blood pressuremonitoring and cGMP studies in vivo.

In vitro vasorelaxation: Thoracic aortae were removed followinganesthesia with intraperitoneal injection of ketamine and xylazine (50and 10 mg/kg, respectively). The paraadventetial tissue surrounding thevessel was carefully removed. Aortae were cut into rings of 4-5 mm andmounted onto the tissue path. The path buffer (Krebs-bicarbonate) wasconstantly gassed with carbogen and maintained at 37° C. The rings werepreloaded under 2 g tension and equilibrated for 90 min with bufferchanged every 15 min. After stabilization, the rings were contractedwith epinephrine (1 μM). Commulative Concentration-Response Curves(CCRC) were constructed for NO-TEMPO, glyceryl trinitrate (GTN) andS-nitroso-N-acetylpenicillamine (SNAP). FIGS. 2 and 3 represent thevaso-relaxant effect of NO-TEMPO as compared to the conventionalNO-donors GTN and SNAP. These figures demonstrate the superiority ofNO-TEMPO to both of these NO-donors.

In vitro induction of GTN tolerance: Aortic rings were treated as forthe control studies except that rings were exposed to 0.44 mM GTN for 1hr. At the end of the tolerance induction period, rings were washedevery 15 min for the following hour. Attempted induction of tolerance toNO-TEMPO was performed under the same conditions as for GTN with ringsexposed to 0.44 mM solution of NO-TEMPO. CCRC were constructed forNO-TEMPO and GTN. FIG. 4 shows the vaso-relaxant effect of GTN on ringsbefore and after exposure to 0.44 mM of the drug and it clearlydemonstrates the development of GTN tolerance under these conditions.This tolerance is also crossed to NO-TEMPO as is obvious from FIG. 5,where a significant difference between the effects of NO-TEMPO on ringsbefore and after pre-treatment with GTN exists. However, when thevaso-relaxant activity of NO-TEMPO and GTN were examined usingNO-TEMPO-pre-treated RARs, no significant differences between thepotencies of these drugs were obtained between control RARs FIG. 2) andNO-TEMPO-pretreated RARs (FIG. 6). This observation demonstrates that,under the condition applied, tolerance to NO-TEMPO does not develop.This conclusion is further supported by separately evaluating thevaso-relaxant effect of each drug on control and NO-TEMPO-pre-treatedRARs. Thus, as is obvious from FIG. 7, no significant difference wasobserved in the vaso-relaxant potency of NO-TEMPO between control andNO-TEMPO-pre-treated RARs. Similarly, no significant difference existsin the potency of GTN between control and NO-TEMPO-pre-treated RARs(FIG. 8). These results demonstrate that, whereas GTN induces toleranceboth to itself and to NO-TEMPO, NO-TEMPO does not induce toleranceneither to itself nor to GTN.

In vivo vasorelaxation and induction of GTN tolerance

Results obtained from evaluating the hypotensive effects of NO-TEMPO invivo also demonstrated the superiority of this drug to both GTN andSNAP. Moreover, vascular cGMP measurements following in vivoadministration of NO-TEMPO showed its superior activity as compared toGTN in control rats and the lack of tolerance induction to its effect inNO-TEMPO pre-treated rats.

In the in vivo experiment, rats were anesthetized with anintraperitoneal injection of a combination of ketamine and xylazine (50and 10 mg per kg, respectively). A catheter was placed in the rightexternal jugular vein and introduced to the level of right atrium. Asecond arterial catheter was placed in the left carotid artery andintroduced to the level of the ascending aorta. The tip of arterialcatheter was connected to a pressure transducer which is connected toanother electrical transducer connected to a computerized Experimetriasystem. Blood-pressure (mean arterial pressure=MAP) was recorded before(basal) and after drug administration. NO-TEMPO was injected through thevenous catheter and induced a significant drop in MAP which lasts for 5to 10 minutes. Values for basal MAP were 130±15 before and 60±10 mm Hgfollowing a 1 mg injection of NO-TEMPO. Unlike with GTN and SNAP, whereattenuation to their effect on MAP develops, the hypotensive effect ofNO-TEMPO on MAP was consistent and was repeatable following successiveadministration of NO-TEMPO, which indicated the lack of tolerancedevelopment to the drug.

Effect on cGMP and Tolerance to Organic Nitrates

Administration of TEMPO-4-mononitrate resulted in a significant increasein cGMP content of vascular tissue. Although it bears only one nitrategroup, the effect of TEMPO-4-mononitrate on vascular cGMP compared wellwith that of GTN, despite the latter containing 3 such groups when bothdrugs are administered intravenously in equimolar doses to control rats.

Experiments were performed in anaesthetized rats as above. Followingi.v. administration of NO-TEMPO (0.1, 0.2, 0.5 mg etc.,) the thorax wasopened and the aorta removed immediately (within thirty seconds from theadministration). After standard processing procedures, cGMP content inthe vascular tissue was measured by Radioimmunoassay (RIA) usingcommercially available kits (i.e., Kit 500 TRK from Amersham). Typicalbasal values of cGMP in aortic tissue were 46±10 versus 197±18 pmole/gtissue obtained following administration of 1 mg of NO-TEMPO (a 4-5-foldincrease in vascular cGMP).

When given subcutaneously (30 mg/kg t.i.d for three consecutive days),NO-TEMPO does not induce tolerance either to itself or to GTN, whereasGTN administered in the same route does induce tolerance to itself andto NO-TEMPO.

Conclusion

It is clear that the compounds of the present invention exhibit bothsuperoxide scavenging and vasodilator activity, fulfilling the combinedroles of organic nitrate and antioxidant. Moreover, they act as potentsuperoxide anion scavengers at concentrations which liberate therapeuticamounts of NO. In addition, the compounds of the present invention donot induce tolerance to themselves or conventional organic nitrates. Thecompounds of the present invention therefore have exceptionaltherapeutic utility, particularly in conditions with adverseresponsiveness to organic nitrates.

What is claimed is:
 1. A compound of the formula:

or a pharmaceutically acceptable salt thereof.
 2. A compositioncomprising the compound of claim 1 and a pharmaceutically acceptableexcipient.
 3. The composition of claim 2, in a form suitable foradministration to a patient, wherein the form suitable foradministration is selected from the group consisting of a form suitablefor topical administration, administration by inhalation, nasaladministration, parenteral administration and oral administration. 4.The composition of claim 3, wherein the composition is in a formsuitable for topical administration.
 5. The composition of claim 4,wherein the composition is in the form of a cream, ointment, gel,aqueous or oil solution or suspension, salve, patch, plaster, lubricantor suppository.
 6. The composition of claim 3, wherein composition is ina form suitable for oral administration.
 7. The composition of claim 6,wherein the composition is in the form of a tablet, capsule, aqueous oroily solution, suspension or emulsion.
 8. The composition of claim 3,wherein the composition is in a form suitable for parenteraladministration.
 9. A composition comprising the compound of claim 1 anda biodegradable polymer.
 10. The composition of claim 9, wherein thepolymer is adsorbed on a stent or applied directly to the site of theangioplasty procedure.
 11. The composition of claim 9, wherein thecomposition is formulated for slow release of the compound.
 12. Acompound of the formula:

or a pharmaceutically acceptable salt thereof.
 13. A compositioncomprising the compound of claim 12 and a pharmaceutically acceptableexcipient.
 14. The composition of claim 13, in a form suitable foradministration to a patient, wherein the form suitable foradministration is selected from the group consisting of a form suitablefor topical administration, administration by inhalation, nasaladministration, parenteral administration and oral administration. 15.The composition of claim 14, wherein the composition is in a formsuitable for topical administration.
 16. The composition of claim 15,wherein the composition is in the form of a cream, ointment, gel,aqueous or oil solution or suspension, salve, patch, plaster, lubricantor suppository.
 17. The composition of claim 14, wherein composition isin a form suitable for oral administration.
 18. The composition of claim17, wherein the composition is in the form of a tablet, capsule, aqueousor oily solution, suspension or emulsion.
 19. The composition of claim14, wherein the composition is in a form suitable for parenteraladministration.
 20. A composition comprising the compound of claim 13and a biodegradable polymer.
 21. The composition of claim 20, whereinthe polymer is adsorbed on a stent or applied directly to the site ofthe angioplasty procedure.
 22. The composition of claim 20, wherein thecomposition is formulated for slow release of the compound.
 23. A methodof treating a condition associated with oxidative stress or endothelialdysfunction comprising administering to a patient in need of suchtreatment a nitric oxide liberating and a superoxide dismutase mimeticeffective amount of a compound of the formula:

a pharmaceutically acceptable salt thereof or a mixture thereof, suchtreatment a nitric oxide liberating and a superoxide dismutase mimeticeffective amount of a compound of the formula:

a pharmaceutically acceptable salt thereof or a mixture thereof.
 24. Themethod of claim 23, wherein the condition is a cardiovascular disease.25. The method of claim 23, wherein the condition is insulin resistancein diabetes.
 26. The method of claim 23, wherein the condition is areproductive disorder selected from the group consisting of penileerection disorder and male impotence.
 27. The method of claim 24,wherein the cardiovascular disease is angina pectoris, atherosclerosis,or hypertension.
 28. The method of claim 24, wherein the methodcomprises administering the compound in conjunction with a stent inangioplasty.
 29. The method of claim 23, wherein the compound isadministered topically, by inhalation, nasally, parenterally or orally.30. The method of claim 23, wherein the method comprises administering acomposition comprising a biodegradable polymer and the compound to thepatient.
 31. A method of treating a diabetic condition associated withoxidative stress or endothelial dysfunction, comprising administering toa patient in need of wherein the condition is selected from the groupconsisting of cardiovascular disease, diabetes, neurodegenerative orneurological disorders, drug and disease-induced nephropathies, andreproductive disorders.
 32. A compound having the formula:

wherein: each R¹ is, independently, hydrogen, alkoyl, alkoxy, carboxy,hydroxy, amino, cyano, nitro, alkyl or a nitric oxide donor group of theformula —X—Y—ONO₂, wherein: X is O, NH, or S; and Y is a C₁₋₂₀ alkylenechain, and wherein at least one R¹ is said nitric oxide donor group andeach R² is alkyl; or a pharmaceutically acceptable salt thereof.
 33. Thecompound of claim 32, wherein each R² is methyl.
 34. A method oftreating a condition associated with oxidative stress or endothelialdysfunction comprising administering to a patient in need of suchtreatment a nitric oxide liberating and a superoxide dismutase mimeticeffective amount of the compound of claim 32, wherein the condition isselected from the group consisting of cardiovascular disease, diabetes,neurodegenerative or neurological disorders, drug and disease-inducednephropathies, and reproductive disorders.
 35. The method of claim 34,wherein the condition is a cardiovascular disease.
 36. The method ofclaim 34, wherein the condition is insulin resistance in diabetes. 37.The method of claim 34, wherein the condition is a reproductive disorderselected from the group consisting of penile erection disorder and maleimpotence.
 38. The method of claim 35, wherein the cardiovasculardisease is angina pectoris, atherosclerosis, or hypertension.
 39. Themethod of claim 35, wherein the method comprises administering thecompound in conjunction with a stent in angioplasty.
 40. The method ofclaim 34, wherein the compound is administered topically, by inhalation,nasally, parenterally or orally.
 41. The method of claim 34, wherein themethod comprises administering a composition comprising a biodegradablepolymer and the compound to the patient.
 42. A method of treating adiabetic condition associated with oxidative stress or endothelialdysfunction, comprising administering to a patient in need of suchtreatment a nitric oxide liberating and a superoxide dismutase mimeticeffective amount of the compound of claim 32.